dTDP-L-鼠李糖生物合成路径的电喷雾多级串联质谱法表征

本研究以2’-脱氧胸苷-5’-二磷酸-L-鼠李糖（dTDP-L-Rha）生物合成路径涉及的系列酶促反应产物为研究对象，建立了电喷雾碰撞诱导解离串联质谱（CID-ESI-MSⁿ）对罕见单糖生物合成路径的表征方法。该方法首先通过ESI-MS技术分析2’-脱氧胸苷-5’-二磷酸-D-葡萄糖-4,6-脱氢酶（RmlB）、2’-脱氧胸苷-5’-二磷酸-4-酮-6-脱氧-葡萄糖-3,5异构酶（RmlC）和2’-脱氧胸苷-5’-二磷酸-4-酮-鼠李糖还原酶（RmlD）酶活产物的形成，随后应用CID-ESI-MSⁿ技术对酶促反应终产物L-鼠李糖（dTDP-L-Rha）的结构进行表征。结果表明：dTDP-L-Rha 的生物合成始于2’-脱氧胸苷-5’-二磷酸-D-葡萄糖（dTDP-D-Glc），依次涉及RmlB、RmlC 和RmlD 三个酶的参与。这与传统的高效液相色谱法（HPLC）监测、纯化制备，核磁共振（NMR）及ESI-MS结构表征的分析结果一致。该方法简单、灵敏、快速，适用于重要罕见单糖生物合成路径的表征。

Characterization of Biosynthetic Pathway of the dTDP-L-Rha by Electrospray Ionization Tandem Mass Spectrometry

Considering the importance of carbohydrate moieties on infectivity and host mimicry, there is a need to better understand the biosynthetic pathways of these unusual sugars in order to identify key targets involved in bacterial pathogenesis. Since the in vitro biochemical characterization of the biosynthetic pathway of unusual sugars is currently hindered by the demand for more accurate, sensitive, and rapid analytical methods for characterizing unusual sugar structures, information about the structure and biosynthesis pathway of these compounds is fragmentary. Mass spectrometry is a rapid, sensitive, and accurate approach for the direct monitoring of enzyme-catalyzed reactions that does not require a chromophore or radiolabeling and thus provides aviable alternative to existing analytical techniques. The objective of this study is to demonstrate the use of electrospray ionization-tandem mass spectrometry (ESI-MS/MS) as a powerful technique for the characterization of enzymatic products in the biosynthetic pathway of deoxythymidine 5’diphosphate-D-rhamnose (dTDP-L-Rha) in E. coliO7. The dTDP-d-glucose-4,6-dehydratase (RmlB), dTDP-4-keto-6-deoxy-glucose-3,5-epimerase (RmlC), and dTDP-4-keto-rhamnose reductase (RmlD) catalyzed reactions were directly monitored by ESI-MS, followed by detailed structural characterization of the final enzymatic products using ESI-MS/MS in the negative-ion mode after minimal cleanup. The biosynthetic pathway of dTDP-L-Rha, beginning from dTDP-L-Rha in three reaction steps catalyzed by RmlB, RmlC, and RmlD, was characterized by ESI-MS/MS. The results obtained were in good agreement with that of traditional high-performance liquid chromatography (HPLC) monitoring and preparation, as well as NMR and ESI-MS structural characterization. Collectively, these data demonstrate that a CID-ESI-MSⁿ based platform is applicable to the facile characterization of the biosynthetic pathway of important unusual dTNP-sugar in the O-chain and offers significant advantages over current methods in terms of speed, sensitivity, reproducibility, automation and reagent costs.